Although many proteins require specific interaction with a ligand for their biological activity, little is known about how such proteins bind ligands or how their synthesis is regulated by ligand availability. To understand assembly of ligand-binding proteins, we are studying the pathway and regulation of bacterial cytochrome biosynthesis. As a model, we are studying cytochrome c2 (cyt c2) of the photosynthetic bacterium Rhodobacter sphaeroides. Cyt c2 is a well-characterized, c-type cytochrome that is essential for photosynthesis. In order to function, the cyt c2 precursor protein (preCycA) must be exported to the periplasm, and bind heme. Our long range goal is to define how and where cyt c2 binds its heme ligand, and determine how cyt c2 synthesis is regulated by heme availability. We seek to answer the answer the following questions regarding cyt c2 biosynthesis. 1. How is heme attachment accomplished? We will determine if heme is attached prior or subsequent to removal of the preCycA signal sequence. The heme lyase enzyme (HeLA) will be purified and used to define the cytochrome and cellular factors required for heme attachment. 2. What cytochrome determinants influence cyt c2 synthesis? We will analyze heme attachment to mutant CycA proteins containing substitutions in the heme binding site and to a series of truncated CycA gene products. In addition, CycA substitutions that block export will be analyzed to see if heme attachment and export are coupled. To determine how individual mutations block cyt c2 synthesis, we will compare the conformation of mutant proteins to the wild type CycA gene product. 3. Does HelA attach heme to more than one cytochrome? We will clone the R. sphaeroides HelA structural gene and construct helA mutants. The phenotype of helA mutants will define whether this enzyme attaches heme to more than one c-type cytochrome. 4. What cycA target sites couple cyt c2 synthesis to ligand availability? Our data suggests the heme precursor delta-aminolevulinic acid (ALA) represses cycA transcription. We will define ALA-responsive cycA promoters, and map the cycA transcription will be determined. 5. Does the repressor interact with target sequences in an ALA-dependent fashion? Transcription from ligand-sensitive cycA promoters appears to be repressed by a protein (ChrR) whose activity requires ALA. Trans-acting mutants in the ALA-responsive repressor will be characterized, and the gene defined by these mutations will be identified. To test if ALA is the physiological co-repressor of cycA transcription, the purified protein will be tested for ALA-dependent binding to cycA promoters.